CD36 Antibody, HRP conjugated

What is CD36 and why is it an important target for immunological studies?

CD36 is a multifunctional glycoprotein receptor expressed on the surface of various cell types including macrophages, platelets, and adipocytes. It serves as a receptor for a broad range of ligands including thrombospondin, fibronectin, collagen, amyloid-beta, oxidized low-density lipoprotein (oxLDL), anionic phospholipids, long-chain fatty acids, and bacterial diacylated lipopeptides . CD36 plays critical roles in lipid metabolism, inflammation, and atherosclerosis, and is involved in the recognition and uptake of various ligands such as oxidized low-density lipoproteins, long-chain fatty acids, and apoptotic cells . Additionally, CD36 is implicated in the pathogenesis of malaria by mediating cytoadherence of Plasmodium falciparum parasitized erythrocytes . Its involvement in multiple physiological and pathological processes makes it a valuable target for researchers studying metabolic disorders, cardiovascular diseases, inflammation, and infectious diseases.

What are the advantages of using HRP-conjugated CD36 antibodies over unconjugated versions?

HRP-conjugated CD36 antibodies offer several methodological advantages:

• Direct detection without secondary antibodies, which simplifies protocols and reduces experimental time

• Elimination of potential cross-reactivity issues associated with secondary antibodies

• Enhanced sensitivity in detection systems using chemiluminescent or colorimetric substrates

• Reduced background in applications where endogenous biotin or other binding sites might interact with detection systems

• Compatibility with multiplexing experiments where multiple primary antibodies from the same host species can be used simultaneously

These benefits make HRP-conjugated CD36 antibodies particularly useful for immunohistochemistry, Western blotting, and ELISA applications where direct detection is advantageous .

What species reactivity should researchers expect when using commercially available CD36 antibodies with HRP conjugation?

The species reactivity of CD36 antibodies varies by manufacturer and clone. Based on the available information:

Researchers should verify species cross-reactivity for their specific application, particularly when working with less common model organisms .

What are the optimal storage conditions for maintaining activity of HRP-conjugated CD36 antibodies?

Storage conditions vary by manufacturer, but general recommendations include:

• Store at 4°C in the dark (Bio-Techne/Novus product)

• Store at -20°C or -80°C upon receipt (ARP product)

• Store at -20°C as received (OriGene product)

The presence of glycerol (noted in some formulations as 50% glycerol) helps prevent freeze-thaw damage. Key practices to maintain antibody performance:

• Avoid repeated freeze-thaw cycles as they can degrade both the antibody and the HRP enzyme

• Store in small aliquots if frequent use is anticipated

• Keep protected from light as HRP is light-sensitive

• Check manufacturer's specific recommendations for preservative content, as some formulations contain preservatives while others don't

• Note the stability period provided by the manufacturer, typically 12 months from receipt under proper storage conditions

How should researchers determine the optimal dilution for CD36 antibody, HRP conjugated for different applications?

Determining optimal dilution requires systematic titration experiments:

• Start with manufacturer's recommended dilution range:

  • For Western blot: Often 1:2000 (OriGene)

  • For IHC: Often 1:50 (OriGene)

  • For other applications: Follow manufacturer's suggestion that "optimal dilutions should be experimentally determined"

• Perform a dilution series experiment:

  • Prepare 3-5 dilutions around the recommended range

  • Include positive controls (tissues/cells known to express CD36)

  • Include negative controls (tissues/cells known to lack CD36 or primary antibody omitted)

• Evaluate signal-to-noise ratio:

  • Optimal dilution shows strong specific signal with minimal background

  • The concentration on the vial label should be consulted if available

For Western blot applications, note that CD36 typically appears at 75-80 kDa despite a theoretical molecular weight of ~53 kDa due to post-translational modifications including glycosylation and palmitoylation .

What positive controls are recommended for validating CD36 antibody, HRP conjugated specificity?

Recommended positive controls include:

• Cell/tissue types with known high CD36 expression:

  • Macrophages

  • Platelets

  • Adipocytes

  • Endothelial cells (CD36 is an endothelial cell marker)

• Specific positive controls mentioned in product information:

  • Antigen standard for CD36 (transient overexpression lysate)

• Validated sample types from literature:

  • Mouse tissues (PMID: 34974159)

  • Rat tissues (PMID: 25635851)

  • Porcine samples (PMID: 23727393)

  • Rabbit tissues (PMID: 30105261)

When validating antibody specificity, researchers should consider both positive controls (expected to show signal) and negative controls (expected to show minimal signal or tissues where CD36 expression is blocked or knocked down).

How can researchers troubleshoot discrepancies between predicted and observed molecular weight of CD36 when using HRP-conjugated antibodies?

CD36 often appears at a higher molecular weight (75-80 kDa) than its predicted size (~53 kDa) in Western blots. This discrepancy is methodologically important to understand:

• Post-translational modifications:

  • CD36 undergoes extensive glycosylation (both N- and O-linked)

  • Palmitoylation at multiple sites affects migration

  • These modifications significantly increase apparent molecular weight

• Troubleshooting approaches:

  • Enzymatic deglycosylation treatments (PNGase F, Endo H) can confirm glycosylation's contribution

  • Sample preparation methods that preserve protein modifications (avoid excessive heating)

  • Use positive control samples with verified CD36 molecular weight

  • Consider tissue/cell-specific variations in post-translational modifications

• Validation strategies:

  • Compare with recombinant CD36 lacking post-translational modifications

  • Use CD36 knockout/knockdown samples as negative controls

  • Consider alternative antibody clones that recognize different epitopes

Researchers should note that the difference between theoretical molecular weight and observed weight on Western blot is expected for CD36 and should not necessarily be interpreted as non-specific binding .

What methodological considerations are important when using CD36 antibody, HRP conjugated for studying its role in lipid metabolism?

When investigating CD36's role in lipid metabolism, several methodological considerations are critical:

• Sample preparation:

  • Careful tissue harvesting to preserve membrane integrity where CD36 resides

  • Appropriate lysis buffers that effectively solubilize membrane proteins without denaturing epitopes

  • Consider subcellular fractionation to differentiate cell surface versus intracellular CD36 pools

• Experimental design:

  • Include physiologically relevant controls (fasted vs. fed state, insulin treatment, etc.)

  • Consider lipid loading experiments to assess CD36 translocation

  • Incorporate metabolic challenges (high-fat diet, fatty acid treatment) to observe dynamic changes

• Detection optimization:

  • For tissues with high lipid content (adipose tissue, fatty liver), additional blocking steps may be necessary

  • Background reduction techniques such as milk-based blockers for Western blots

  • Additional washing steps for immunohistochemistry applications

• Functional correlation:

  • Combine CD36 detection with functional assays of fatty acid uptake

  • Consider dual labeling with lipid droplet markers or other metabolic proteins

  • Correlate CD36 localization/expression with physiological or pathological endpoints

CD36 functions as fatty acid translocase and is involved in long-chain fatty acid uptake, making these considerations particularly important for metabolism-focused research .

What are the best practices for using CD36 antibody, HRP conjugated in examining its role in atherosclerosis and inflammation?

When investigating CD36's role in atherosclerosis and inflammation:

• Cell-specific expression analysis:

  • Use dual labeling approaches to identify CD36-expressing cell types in lesions

  • Compare expression across different vascular beds and lesion stages

  • Correlate with markers of inflammation (cytokines, adhesion molecules)

• Ligand interaction studies:

  • Examine co-localization with oxidized LDL in atherosclerotic plaques

  • Assess binding of other CD36 ligands (thrombospondin, amyloid-β, etc.)

  • Consider competitive binding assays to determine relative affinity

• Technical considerations:

  • Careful fixation methods that preserve both CD36 epitopes and lipid components

  • Antigen retrieval optimization for formalin-fixed tissues

  • Signal amplification methods for detecting lower expression levels

• Experimental models:

  • Compare CD36 expression between normal and atherosclerotic vessels

  • Analyze inflammatory stimuli effects on CD36 expression

  • Consider time-course experiments to track CD36 expression changes during disease progression

• Controls and validation:

  • Include appropriate disease controls (normal vs. pathological samples)

  • Use validated tissue sections from previous publications

  • Consider genetic models (CD36-deficient) as negative controls

CD36 acts as a scavenger receptor that binds oxidized LDL and plays a significant role in foam cell formation during atherosclerosis development, making these methodological approaches particularly relevant .

How can researchers optimize dual labeling experiments involving CD36 antibody, HRP conjugated and other markers?

Optimizing dual labeling experiments with HRP-conjugated CD36 antibodies requires careful planning:

• Sequential detection approach:

  • Complete the HRP-conjugated CD36 antibody detection first

  • Inactivate HRP using hydrogen peroxide (3% H₂O₂ in PBS for 10 minutes)

  • Perform second antigen detection with an alternative system (alkaline phosphatase, fluorescence)

  • Validate complete inactivation through control experiments

• Spectral separation strategies:

  • Use spectrally distinct substrates for different enzymes

  • For colorimetric detection, pair HRP (brown/DAB) with AP (red/Fast Red)

  • For chemiluminescence, use fluorescent substrates with non-overlapping spectra

• Antibody selection considerations:

  • Choose primary antibodies from different host species when possible

  • Validate each antibody individually before dual labeling

  • Perform absorption controls to check for cross-reactivity

• Order of application:

  • Test different sequences of antibody application to determine optimal protocol

  • Generally apply antibodies against less abundant targets first

  • Consider antigen masking effects when epitopes are in close proximity

• Signal amplification balance:

  • Adjust dilutions of each antibody to achieve comparable signal intensities

  • Consider tyramide signal amplification for the weaker signal

  • Establish appropriate exposure times for each signal individually

These approaches enable researchers to effectively study CD36 in relation to other markers in the same sample, providing valuable co-localization data .

What fixation methods are optimal for preserving CD36 antigenicity in immunohistochemistry when using HRP-conjugated antibodies?

Fixation methods significantly impact CD36 detection in tissue sections:

• Formalin fixation considerations:

  • Standard 10% neutral-buffered formalin is compatible with CD36 detection

  • Limit fixation time (24-48 hours optimal, longer fixation may require stronger antigen retrieval)

  • Post-fixation processing (alcohol dehydration, paraffin embedding) should follow standard protocols

• Frozen section advantages:

  • Minimal epitope masking makes frozen sections ideal for CD36 detection

  • Light fixation (4% paraformaldehyde for 10-15 minutes) preserves morphology while maintaining antigenicity

  • Consider acetone fixation (10 minutes at -20°C) as an alternative that works well with CD36 detection

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is often effective

  • For stronger retrieval, consider EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 10-20 minutes) based on fixation duration

• Comparison of methods:

  Fixation Method	Advantages	Disadvantages	Recommendation
  10% NBF/Paraffin	Good morphology, long-term storage	Requires antigen retrieval	Standard approach, validated in literature
  Frozen sections	Excellent antigen preservation	Poorer morphology	Best for co-localization studies
  Acetone fixation	Quick, good for membrane proteins	Limited morphology preservation	Good for cells and small tissue pieces

Multiple commercially available CD36 antibodies with HRP conjugation have been validated for both frozen and paraffin-embedded sections .

How can researchers minimize background issues when using CD36 antibody, HRP conjugated in tissues with high lipid content?

Tissues with high lipid content (adipose tissue, fatty liver, atherosclerotic plaques) present special challenges for CD36 immunostaining:

• Enhanced blocking protocols:

  • Extended blocking times (1-2 hours at room temperature)

  • Use of specialized blockers containing both proteins and detergents

  • Sequential blocking with different blockers (e.g., 5% BSA followed by 10% normal serum)

  • Addition of 0.1-0.3% Triton X-100 to improve penetration through lipid-rich areas

• Modified washing procedures:

  • More frequent and longer washing steps

  • Include 0.05-0.1% Tween-20 in wash buffers

  • Consider using PBS with higher salt concentration (PBS + 0.5M NaCl) for one wash step

• Antibody diluent optimization:

  • Dilute antibodies in solution containing 1-2% BSA and 0.05% Tween-20

  • Consider adding 5-10% normal serum from the same species as the tissue

  • Low concentration of non-ionic detergents helps reduce hydrophobic interactions

• Endogenous peroxidase quenching:

  • More thorough quenching (3% H₂O₂ for 15-20 minutes)

  • For highly vascular tissues, consider using H₂O₂ in methanol rather than aqueous solution

  • Perform quenching step before blocking for better penetration

• Control sections:

  • Include isotype controls at the same concentration as primary antibody

  • Process serial sections with primary antibody omitted

  • Consider absorption controls with immunizing peptide when available

These approaches help distinguish true CD36 signal from background issues in challenging tissues with high lipid content .

What are the recommended approaches for quantifying CD36 expression levels using HRP-conjugated antibodies in Western blot applications?

Accurate quantification of CD36 in Western blot applications requires methodological rigor:

• Sample preparation optimization:

  • Standardize protein extraction methods across samples

  • Include appropriate protease inhibitors to prevent degradation

  • Avoid excessive heating which can cause aggregation of membrane proteins

  • Consider membrane fraction enrichment for enhanced CD36 detection

• Quantification controls:

  • Include a concentration gradient of recombinant CD36 for standard curve generation

  • Use housekeeping proteins appropriate for the experimental context

  • For membrane proteins, Na⁺/K⁺-ATPase or calnexin may be better controls than cytoskeletal proteins

  • Include positive control samples with known CD36 expression levels

• Technical considerations:

  • Ensure equivalent protein loading (20-50 μg total protein typically sufficient)

  • Transfer efficiency monitoring with reversible staining (Ponceau S)

  • Optimize exposure time to ensure detection within linear range

  • Use digital image acquisition systems rather than film for better quantification

• Data analysis approaches:

  • Express CD36 levels relative to appropriate loading controls

  • For comparing across multiple blots, include a common reference sample on each blot

  • Consider normalization to total protein staining methods (SYPRO Ruby, Coomassie)

  • Statistical comparison of replicate experiments (minimum n=3)

• Validation considerations:

  • Verify specificity with known positive and negative controls

  • Consider alternative detection methods (ELISA, flow cytometry) for confirmation

  • Account for the higher molecular weight (~75-80 kDa) of CD36 due to glycosylation

These methodological approaches enable reliable quantitative comparison of CD36 expression across experimental conditions .

How can CD36 antibody, HRP conjugated be used to investigate the role of CD36 in pathogen recognition and infectious disease models?

CD36 plays important roles in pathogen recognition and infectious disease processes that can be studied using HRP-conjugated antibodies:

• Malaria research applications:

  • Detect CD36 expression on endothelial cells and correlate with cytoadherence of Plasmodium falciparum-infected erythrocytes

  • Examine CD36 localization at the interface between infected erythrocytes and host cells

  • Monitor changes in CD36 expression during infection progression

  • Investigate co-localization with other receptors involved in parasite sequestration

• Bacterial infection studies:

  • Analyze CD36 expression in response to bacterial diacylated lipopeptides

  • Study CD36-dependent phagocytosis of bacteria by macrophages

  • Investigate CD36 clustering and signaling in response to bacterial components

  • Correlate CD36 expression with bacterial clearance in tissue samples

• Technical considerations:

  • Use appropriate fixation protocols that preserve both CD36 epitopes and pathogen structures

  • Consider dual labeling approaches to visualize CD36-pathogen interactions

  • Include time-course analysis to track dynamic changes in CD36 expression

  • Implement quantitative image analysis for co-localization studies

• Experimental validation:

  • Compare results between in vitro and in vivo infection models

  • Use CD36 blocking antibodies or knockdown approaches as functional controls

  • Include genetically modified pathogens lacking CD36-binding ligands

  • Correlate immunohistochemistry findings with functional assays of pathogen adhesion

The direct HRP conjugation enables more precise localization studies with reduced background, which is particularly valuable when examining the interface between host cells and pathogens .

What considerations are important when using CD36 antibody, HRP conjugated for studying CD36 in the context of neurodegenerative diseases?

CD36's roles in neuroinflammation and amyloid-β interactions make it relevant to neurodegenerative disease research:

• Brain tissue-specific considerations:

  • Optimize fixation and antigen retrieval for preservation of both CD36 epitopes and neural structures

  • Consider post-fixation times carefully as brain tissue may require different processing

  • Use appropriate permeabilization methods for improved antibody penetration

• Cell-specific expression analysis:

  • Implement dual labeling with cell-type specific markers (microglia, astrocytes, neurons)

  • Analyze regional variations in CD36 expression across brain structures

  • Compare expression patterns between healthy and diseased tissues

• Amyloid-β interaction studies:

  • Investigate co-localization between CD36 and amyloid deposits

  • Examine relationship between CD36-expressing cells and plaque formation

  • Study temporal relationship between CD36 upregulation and disease progression

• Technical optimization:

  • Reduce autofluorescence through specialized treatments (Sudan Black B)

  • Consider signal amplification methods for detecting low expression levels

  • Implement careful blocking of endogenous peroxidase in highly vascular regions

  • Use antigen retrieval methods optimized for fixed brain tissue

• Functional correlations:

  • Link CD36 expression patterns to inflammatory markers

  • Correlate with indicators of phagocytic activity

  • Examine relationship to disease severity markers

CD36 functions as a receptor for amyloid-β and contributes to microglial inflammatory responses in neurodegenerative contexts, making these methodological considerations particularly relevant .